Hematopoietic stem cells are a rare population of cells which have the unique potential to aid cellular therapy for hematological malignancies, bone marrow failure disorders, as well as gene therapy. These cells are most commonly isolated using the CD34 antigen. CD34 is expressed specifically by these cells, but its function is unknown. Whether CD34 function plays a role in stem cell biology is an important question. Furthermore, CD34 expression on the earliest stem cell remains uncertain, although it is well-documented that more mature stem cells express CD34. However, it may be critically important in diseases with stem cell intrinsic defects to use the most primitive stem cell for cellular therapy. Our first specific aim will examine the role for CD34 expression for self-renewal, proliferation, and differentiation of primitive stem cells. This will involve transplantation of CD34 null stem cells into normal mice and normal cells into null mice to test the competence of these null cells. In this setting we will also examine the recovery of endogenous CD34 null stem cells to either radiation of 5-fluorouracil exposure. Specific aim 2 will study the compensatory mechanisms that may be at play in allowing CD34 null mice to survive to adulthood with mildly affected hematopoietic phenotypes. Specifically, cells present in the normal thymus have been shown to support or inhibit hematopoiesis. We will determine if these thymic cells in CD34 null mice provide the same functions. In this aim we will also study the homing potential of stem cells from CD34 null mice to engraft normal recipients using a recently developed homing assay in which we will tag stem cells in vivo. In specific aim 3 will decipher the mechanism by which CD34 blocks the differentiation of hematopoietic cells. We will identify protein(s) which interact with the important regulatory domain within the intracellular region of the CD34 molecule that may negatively control differentiation. We expect that the results of our studies will be directly applicable to treatment of hematological disorders, genetic diseases, and malignancy.